The present invention relates to a purified plant cytokinin oxidizing enzyme (ckx1) from Zea mays, the complete amino acid sequence of which has been elucidated, and to isolated nucleotide sequences encoding the enzyme. The invention further relates to novel methods for moderating the concentration of the enzyme and similar enzymes in plants in order to affect plant cell growth and death. Applications of the invention include the regulation of the production of ckx1 in plant roots to affect pathogenesis, the regulation to alter plant habit, and the bulk production of ckx1 enzyme for use in a plant biochemical assay.
Plant cytokinins are a class of plant hormones which, when combined with auxin, control cell division, promote shoot development from callus, release lateral buds from dormancy, and regulate plant structure and growth in a variety of ways. The naturally occurring active cytokinins in most higher plants are free-base zeatin (6-(4-hydroxy-3-methylbut-trans-2-enylamino)purine) (hereinafter Z), and its 9-riboside (hereinafter ZR). Plant tissues normally contain, therefore, Z, ZR, and smaller amounts of N.sup.6 -(.DELTA..sup.2 -isopentenyl)adenine (hereinafter, iP) derived from biosynthetic precursors. Elevated cytokinin levels are associated with the development of seeds in higher plants, and have been demonstrated to coincide with maximal mitotic activity in the endosperm of developing maize kernels and other cereal grains. Exogenous cytokinin application (via stem injection) has been shown to directly correlate with increased kernel yield in maize. In addition, plant cells transformed with the ipt gene from Agrobacterium tumefaciens (encoding a dimethylallylpyrophosphate:5'-AMP transferase capable of increasing cellular production of Z and ZR) showed increased growth corresponding to an increase in endogenous cytokinin levels upon induction of the enzyme. Thus, given the biosignificance of cytokinins to the growth of plants, the ability to manipulate cytokinin levels in higher plant cells is of great commercial and scientific interest.
The action of cytokinin oxidase is a major method of effective cytokinin catabolism in plant cells. This inactivation of cytokinin is accomplished by the oxidative removal of the side chain from cytokinin free bases (or their ribosides) in the presence of molecular oxygen. An example of this reaction with iP is shown in FIG. 1a. Although the exact chemical mechanism for this reaction is unknown, it is suspected that the enzyme is reduced during the deprotonation of iP to N.sup.6 -(.DELTA..sup.2 -isopentenylimino)purine. The purine is then hydrolyzed into adenine and intermediate 3-methyl-2-butenal (FIG. 1b).
While the electron acceptor responsible for reoxidizing the reduced enzyme in plant cells is not known, molecular oxygen can do so in vitro. Alternatively, the reduced enzyme may be reoxidized in vitro by intermediates such as Cu.sup.+2 /imidazole complexes or the artificial electron acceptor dichlorophenolindophenol (DCPIP).
Cytokinin oxidases are known to remove cytokinins from plant cells after cell division, and have also been postulated to be involved in the senescence process. Cytokinin oxidase activities have been shown to positively correlate to the mitosis of endosperm cells in maize kernels, along with the increase in natural cytokinin concentrations. Oxidase activity increases shortly after the increase in endogenous cytokinin levels. A similar correlation was demonstrated with artificially increased cytokinin levels in transgenic tobacco. Thus, expression of cytokinin oxidases is thought to be involved in the maintenance of hormonal homeostasis in developing plant cells. Because cytokinin oxidases appear to be substrate-inducible, they act in a negative regulatory fashion to reduce elevated cytokinin levels back to basal values. This substrate induction of cytokinin oxidase activity is a significant barrier to potential commercial applications which attempt to manipulate cytokinin levels in transgenic plants through increased cytokinin production.
Cytokinin oxidases have been discussed for a number of plant species, including Vinca rosea, beans (Phaseolus vulgaris and lunatus), wheat (Triticum aestivum), tobacco (Nicotiana tabacum), Dianthus caryophyllus, soy (Glycine max), and maize (Zea mays). All of these plant cytokinin oxidases have a similar substrate preference for iP and Z, but show limited or no reactivity with bulky, reduced, or aromatic side chain cytokinins. All also exhibit enhanced activity in the presence of copper plus imidazole. However, these enzymes show substantial variation in both specific activity and molecular weight. This is thought to be linked to the occurrence of glycosylated and unglycosylated variants of the protein, both between and within species.
In the case of the glycosylated cytokinin oxidase, the heavily glycosylated protein may present a carbohydrate-rich surface, preventing antibody formation against peptide epitopes. The glyco-epitopes to which antibodies are raised under these conditions are non-specific, and may prevent isolation of the protein, or clones containing the gene which encodes it, via immuno-chromatography or other immunology-based means. An earlier reported attempt to isolate the gene for maize cytokinin oxidase (ckx1) by immunoscreening of maize cDNA library expression products (Burch, 1992) was unsuccessful.
As demonstrated, the full amino acid sequence and encoding DNA for a cytokinin oxidase has been a long sought after goal in modern plant physiology.